In one aspect, the present invention relates to fungal growth media comprising oxgall, purified saponin, a substrate for phenol oxidase and a supporting agent therefor, such as agar, for example. In another aspect, this invention relates to a differential medium for the detection and identification of pathogenic yeast-type fungi. In still another aspect, the present invention relates to storable dry fungal media preparations which, upon addition of water, provide growth media useful in a rapid differential analysis for several of the more common pathogenic fungi present in body fluid samples. In still a further aspect, the fungal media of the present invention provide an especially rapid and reliable technique for the detection of, and differentiation of, two medically important fungi, Candida albicans and Cryptococcus neoformans.
Under normal conditions, various types of yeasts (that is, single cell fungi) will be present in the body as saprophytes and will not interrupt normal body functioning because the fungal growth will be controlled by normal types of bacteria which are also present. However, when a patient is treated for bacterial infections, through the use of antibacterial antibiotics for example, the normal bacterial-fungal balance will be disrupted and fungal infection becomes a real possibility. Cancer patients, because of the multiple antibacterial drugs administered and because of an incompetent immune system, are especially susceptible to fungal infection. Fungal infection can cause a rapid deterioration of the patient's condition, especially if the patient is already in a weakened state resulting from bacterial infections, for example. Such fungal infections can precipitate serious conditions, for example, meningitis.
Two of the most medically important types of fungi are Candida albicans and Cryptococcus neoformans. Candida albicans is a saprophyte found in the gastrointestinal tract of man. Under certain conditions, however, this yeast may become invasive causing a severe and usually fatal disease in the debilitated patient. Cryptococcus neoformans is especially important because of its predilection for the central nervous system which can cause severe disease in a biologically defenseless patient.
The presumptive identification of Candida albicans depends solely upon morphological changes which occur when this fungus is plated and allowed to grow on an appropriate medium. The first morphological change indicative of the presence of Candida albicans is the formation of germ tubes, which appear as tiny appendages extending from the plated unicellular specimens. These germ tubes eventually grow into elongated filaments extending outwardly from the body of the Candida albicans. Formation of germ tubes within 2 to 3 hours after plating of the fungus is presumptive evidence that Candida albicans is present. In a second stage of growth, generally round bodies appear at the ends of the filaments. These round bodies are known as chlamydospores. Only two species of the Candida genus will form chlamydospores. These are Candida albicans and Candida stellatoidea. Thus, chlamydospore formation is indicative of the presence of either Candida albicans or Candida stellatoidea.
Identification of Cryptococcus neoformans is generally recognized to be more difficult than the identification of Candida albicans in that Cryptococcus neoformans undergoes no morphological changes which can be observed and remains unicellular throughout its growth patterns. Until recently, one or more of three basic tests, or a combination thereof, were employed to identify the presence of the genus Cryptococcus. One of the methods of identification comprises microscopic inspection of a specimen to identify whether or not a capsule-like formation around the cells of the fungi is present. In order to aid in the inspection of such capsule-like formations, a specimen is surrounded with india ink which enhances the appearance of the capsule by providing a clear and translucent image against the black background making such capsules easier to identify during microscopic examination. A second method employed to identify the genus Cryptococcus comprises plating the specimen in a medium containing urea and a color indicator. Because Cryptococcus neoformans produces an enzyme known as urease it has the capability to break down and use the nitrogen contained in the urea, causing the pH to rise, thereby changing the color of the indicator. Therefore, growth on a urea containing medium is indicative of the presence of Cryptococcus neoformans. A third method for the identification of the genus Cryptococcus relies on the ability of that genus to produce a starch-like compound. When the starch-like compound is present addition of iodine will cause a purple ring to appear around the colony. It is to be noted that neither of these tests is specific for Cryptococcus neoformans by itself or in combination, as other species within the genus Cryptococcus and other genera of yeasts may also give a positive reaction.
Therefore, in order to identify the species Cryptococcus neoformans additional tests have had to be employed in the past. These include the development of a growth profile of a specimen when plated on a series of carbohydrate containing media. For example, up to fourteen different types of carbohydrates can be incorporated into growth media and after 7 to 21 days a specimen plated thereon will develop a growth profile which will indicate whether or not Cryptococcus neoformans is possibly present in the specimen. Another method for identifying the species is to plate the fungal sample on an agar medium containing creatinine and if significant growth is observed within 5 to 8 days such growth is indicative of the presence of Cryptococcus neoformans because of that fungi's capability to assimiliate the creatinine. However other genera of yeast can also grow on creatinine so this test is not specific for Cryptococcus neoformans.
Perhaps the single most successful and specific conventional test for Cryptococcus neoformans includes the use of bird seed agar. It was discovered that if Cryptococcus neoformans was present in a sample plated on bird seed agar a specific tell-tale brown color would appear, as the specimen grew on the plate, within a period of five days to two weeks. This method was improved by employing an extract of bird seed which lowered the identification time 3 to 5 days. Later it was discovered that the brown pigment coloration was the result of the reaction between the enzyme phenol oxidase and a particular substrate present in bird seed agar. Accordingly, use of substituted phenols such as caffeic acid in the growth medium further shortened the period of time necessary for identification to about forty-eight hours. A still further refinement of the use of caffeic acid to identify Cryptococcus neoformans is set forth in an article by Hopfer and Groschel entitled "Six Hour Pigmentation Tests for the Identification of Cryptococcus Neoformans", Journal of Clinical Microbiology, August 1975, Vol. II, No. 2, p. 96-98. The improvement set forth therein includes combining caffeic acid with ferric citrate and incorporating these compounds onto paper discs for use as substrates for the phenol oxidase enzyme activity of Cryptococcus neoformans. Use of these caffeic acid-ferric citrate impregnated paper discs further lowered the identification time to 3 to 6 hours. However, the solution of caffeic acid and ferric citrate used to impregnate the paper discs is quite unstable when exposed to light and therefore presents serious storage problems. Furthermore, the relative concentrations of caffeic acid and ferric citrate are critical and an unbalanced combination will require longer incubation periods for production of a dark pigment, or, in some cases, nonspecific pigmentation of saprophytic Cryptococcus species and several Candida species. Furthermore, while identification times are lowered to 3 to 6 hours from the time of plating onto the paper discs the sample must be grown on a "primary" medium before plating onto the ferric citrate, caffeic acid impregnated discs.
It should be noted that, while Candida albicans and Cryptococcus neoformans are two of the most medically important yeasts, no conventional test procedure or medium can be employed to simultaneously identify the presence of both of these fungi. Instead, it has been necessary in the past to run two tests, one for the presence of Candida albicans and a second test, such as one of those described above, to identify Cryptococcus neoformans.
Recently a new culture medium for the identification of Cryptococcus neoforms, Candida albicans and Candida stellatoidea was discovered which includes caffeic acid, oxgall and an emulsifying agent sold under the tradename Tween 80 by Atlas Chemical Company. This medium is described by Fleming, Hopkins and Land in an article entitled, "New Culture Medium for the Presumptive Identification of Candida albicans and Cryptococus neoformans," Journal of Clinical Microbiology, February 1977, Vol. V, No. 2, p. 236-243 and provides for the specific identification of both Candida albicans and Cryptococcus neoformans. While identification of Cryptococcus neoformans is slower than when the caffeic acid-ferric citrate impregnated paper discs discussed above are employed the Tween 80, oxgall, caffeic acid medium (hereinafter sometimes referred to as TOC) has certain advantages in that it is not light sensitive and the concentration of components do not have to be as critically formulated as the ferric-citrate discs. The only disadvantage of the TOC medium is that Candida krusei, a filamenting yeast, will not form pseudohyphae. Thus, while the presence of Candida albicans or Candida stellatoidea can be determined by the formation of germ tubes within 3 hours of plating, the presence of Candida krusei may remain undetected as no filamentation appears even after 24 hours on TOC medium. Further, while the shelf life of prepared TOC medium is good (about 2 to 3 weeks) a longer shelf life is, of course, desirable. In addition, because Tween 80 is a liquid composition, TOC medium does not lend itself to the formulation of a completely dehydrated dry powder composition easily storable in powdered form for preparation and use as needed. This medium does, however, provide for a single test procedure which will identify the presence of Candida albicans, Candida stellatoidea and Cryptococcus neoformans.
Thus, while a variety of methods and media have been employed in order to identify and differentiate various fungal pathogens including the important species Candida albicans and Cryptococcus neoformans, there is a continuing need for fungal growth media which will rapidly identify and differentiate those species as well as other opportunistic fungi and which are relatively easy to prepare and use and which have a relatively long shelf life.